Optimizing Preconditions for More Consistent Starting Potentials

Preconditions were used to reverse the black spot formation that occurred when inserting the pipette into the solution and before a bias was applied. They were also used to make a system more consistent to influence transformation times to be more reproducible. After performing more experiments, it was noticed that performing consecutive experiments provided different starting responses. Here we are attempting to change the precondition parameters to eliminate the inconsistencies between multiple experiments. The experiments that used a constant bias, shown later, shows the inconsistencies between multiple experiments. The parameters for the

Figure 3-15 Consecutive experiments with inconsistent precondition results. This is a voltage response curve showing 2 different experiments, Run 1 and Run 2. Run 1 is identified as the black curve and run 2 is identified as the red curve. The blue arrow points from the precondition sections of both curves and points to a magnified version of the same curves. The color code still applies in the magnified view. This experiment used a 200 nm pipette, 1 M HCl, 7.9 pH insulin solution, 0.18 mm Ag/AgCl working electrode.

Figure 3.15 shows the voltage response curve with 2 different experiments. Both

experiments use the same nanopipette and the same conditions. These experiments were taken in sequence. Different potential responses are obtained for both runs. In the magnified view, it can be observed that the first run’s potential after precondition was -580 mV and the second run had a potential of -448 mV. There is a 132 mV difference between the first and second experiments. The starting potentials, time = 0, are -360 mV for the run 1 and -247 mV for run 2, which

provides a 113 mV difference. This deviation between runs is something that could directly affect consistency. This appears to be caused by using a constant current precondition.

The precondition does not have to be a constant current or potential, it can take on different waveforms e.g. sine, triangular, square. The duration and magnitude can also be changed as shown in Figure 3.16

Figure 3-16 Different Alternate Precondition Waveforms. A) A graph displaying a section of the applied current conditions using a triangular waveform with a 10 nA bias and 1 Hz frequency. B) An applied current graph that uses a sine waveform with a 10 nA bias and 1 Hz frequency. C) A preconditioning experiment showing the applied current for a duration of 30 seconds applying 10 nA.

Figure 3.16 demonstrates some of the options other than a constant bias being applied to the system. A system can use a constant applied bias; however, it can also use an alternating

waveform such as a triangular or sine wave. We use different waveforms to test the effect on consistency and preventing black spot formation.

Fig. 3.16 A shows a triangular waveform being applied, using an amplitude of 10 nA at 1 Hz. Both the frequency and amplitude of the alternating current waveform can be adjusted.

Fig. 3.16 B displays a sine wave form that is applied to the system. This waveform applies a 10 nA peak bias and a 1 Hz frequency. The amplitude and the frequency can be changed along with duration of the the precondition.

Fig. 3.16 C illustrates an example of an experiment to test preconditions. This example applies a sine wave for 30 seconds. It applies 10 nA peak current at 1 Hz frequency. The duration for the experiment can be increased or decreased. Just like all of the waveforms shown in Figure 3.16 the amplitudes can be increased or decreased along with increasing or decreasing the frequency.

Figure 3-17 Using Alternating Current Preconditions to provide more reproducible starting potentials. The top half consists of a sine wave precondition. A) This is the applied current using a sine wave. B) This is the voltage response of the applied current that used a sine waveform. The bottom half consists of a triangular precondition. C) This is the applied current using a triangular wave. D) This is the voltage response of the applied current that used a triangular waveform.

Figure 3.18 displays four different curves that show the examples of two different alternating current waveforms, triangular and sine. The top half of the figure has two curves that represent the sine waveforms. There are insets added to each section displaying the waveform applied, for clearer viewing. The bottom half of the figure has two curves that represent the triangular waveform. These experiments use the same nanopipette and the same internal and external solution. The two experiments conducted were performed sequentially.

Fig 3.18 A is the applied current using a sine waveform at 10 nA held for 30 seconds at 1 Hz. The conditions frequency, amplitude, and duration can be adjusted.

Fig 3.18 B is the voltage response curve that was the made by Fig 3.18 A. It can be observed that there is a drift due to polarization from the positive bias being applied. The point of interest is where the alternating current application ends, indicated by the red dot. The other point used for analysis is the start of the experiment at t = 0. We can compare these points after running multiple experiments to see if by applying an alternating current waveform if we can achieve a more consistent preconditioning treatment before a ramp or maximum bias is applied.

Fig 3.18 C is the applied current using a triangular waveform which is held at 10 nA for 30 seconds at 1 Hz. Again these variables can be adjusted.

Fig 3.18 D is the voltage response curve that was the made by Fig 3.18 C. It can be observed that there is a drift due to polarization from the positive bias being applied. The point of interest is where the alternating current application ends, indicated by the red dot.

The observations made from conducting these experiments were that applying triangular waveforms were not as effective for reversing the black spot formation. We found that the triangular waveform would not provide a more consistent starting potential before applying the positive bias to the system for nucleation. Sine waveform was more successful for reversing and/or shrinking the black spot. The amplitudes tested were 500 mV and 100 mV in both the positive and negative polarities. Applying a negative bias greatly reduced black spot size to the scale where it was not visible. Applying a positive bias did not reverse the black spot. When testing the -100 mV and -500 mV a difference in reversing the black spot was not noticeable. The frequencies were also tested using a 10 Hz and a 1 Hz frequency. There was not a noticeable difference in reversing the black spot. While we found alternative ways to reverse the black spot

formation did not find a meaningful way to improve consistent starting potentials between consecutive experiments.